NKT cells offer an enormous therapeutic potential if we learn how to manipulate them in vivo. As cellular adjuvants of immunity, NKT cells prime and control the maturation of dendritic cells in al infectious contexts. As such, the recruitment of these cells could be critical to vaccine development, immunotherapy of chronic infectious diseases, prevention of infection, and cancer vaccines. However, success will rely heavily on our ability to translate the knowledge of mouse NKT biology to humans. For instance, the pharmacology of compounds capable of activating NKT cells is almost unexplored as are cellular uptake and catabolism. Since, activators of NKT cells are glycolipids, it is most likely that lipoprotein and scavenger receptor polymorphism in human will heavily influence the potency of this new family of immunomodulators. We propose to address some fundamental aspects of the molecular pharmacology of NKT agonists in mice and humans to help a successful use of those molecules in medicine. This goal will be attained through 3 specific aims: Aim 1: Proteomic studies of NKT cell agonist transport and metabolism. Based on our previous work, we hypothesize that NKT agonists have a unique biology when compared to other lipids. Original tools need to be designed and used in the context of NKT biology to drive substantial progress in the field and its translation to clinical studies. We will combine novel chemistry and recombinant protein engineering to access the molecular pharmacology of NKT agonists. Aim 2: Transport of NKT cell agonists. Using traditional serum biochemistry, we have isolated and characterized the function of the association between FAAH and GalCer. Using proteomic approaches we have now profiled serum for all GalCer-associated proteins. We hypothesize that many of these proteins will impact positively or negatively the biology of NKT agonists. The study of each of these proteins will be carried out using a robust experimental system combining recombinant protein expression, in vitro biophysical and functional studies, and in vivo model systems of vaccination before being translated to human cells. Aim 3: Cellular uptake and catabolism of NKT cell agonists. Uptake, processing and catabolism of NKT agonists have been only superficially examined. We will combine proteomics studies with novel biochemistry and cell biology approaches to understand the delivery and processing of NKT antigens to CD1d loading compartments. As NKT agonists are entering rapidly the clinical field, the knowledge that we will acquire through the current proposal will be critical for successful translational studies.